Human leukocyte interferons designated .alpha..sub.1, .alpha..sub.2, .beta..sub.1, .beta..sub.2, .beta..sub.3, .gamma..sub.1, .gamma..sub.2, .gamma..sub.3, .gamma..sub.4, and .gamma..sub.5 derived from virus induced normal or leukemic donors' leukocytes have been purified to homogeneity, see U.S. Pat. No. 4,289,690, issued Sept. 15, 1981. More recently, recombinant DNA technology has been employed to occasion the microbial production of a number of different leukocyte interferons amino acid sequences exhibit on the order of 70 percent or more homology, one relative to another as disclosed in U.S. Patent Application Ser. No. 205,578, filed Nov. 10, 1980, now abandoned, Inventors Goeddel and Pestka. Specific recombinant human leukocytes described in this application are IFL-rA, B, C, D, F, G, H, I and J. Additionally, U.S. Patent Application Ser. No. 305,657, filed Sept. 25, 1981, now U.S. Pat. No. 4,456,748, Inventor, Goeddel, describes the microbial production via recombinant DNA techology of hybrid leukocyte interferons. Examples of such hybrid leukocyte interferons include IFLr A.sub. 1-91 D.sub.93-166.D.sub.1-92 A.sub.92-165,A.sub.1-62 D.sub.64-166 D.sub.1-63 A.sub.63-165.A.sub.1-91 B.sub.93-166,A.sub.1-91 F.sub.93-166, A.sub.1-91,G.sub.93-166,A.sub.1-150 I.sub.151-165,B.sub.1-92 A.sub.92-165 B.sub.1-92 D.sub.93-166,B.sub.1-92 F.sub.93-166,B.sub.1-92 G.sub.33-166, D.sub.1-92 B.sub.93-166,D.sub.1-92 F.sub.93-166,D.sub.1-92 G.sub.93-166, F.sub.1-92 A.sub.92-165,F.sub.1-92 B.sub.93-166,F.sub.1-92 D.sub.93-166, F.sub.1-92 G.sub.93-166, and I.sub.1-151 A.sub.152-166.
The aforesaid natural, recombinant and hybrid human leukocyte interferons represent a family of proteins characterized by a potent ability to confer a virus-resistant state in their target cells. In addition, these interferons can act to inhibit cell proliferation and modulate immune response. These properties have prompted the initial clinical use of IFLrA and IFLrD as therapeutic agents for the treatment of viral infections and malignancies.
Crystallization of a substance satisfies one of the classical criteria for homogeneity. Additionally, the crystallization process itself can provide a useful purification step. The availability of large ordered crystals of human leukocyte interferons will also allow the determination of the molecule's tertiary structure utilizing X-ray crystallization.
Numerous techniques have been developed for the crystallization of proteins, however, no generalized procedure has been discovered, and many proteins remain uncrystallized. Thus, crystallization of proteins is an unpredictable art utilizing trial and error procedures among many possible alternative methodologies.
One of the most widely used approach involves the addition to the protein solution of a crystallizing agent, which is commonly a salt, such as ammonium sulfate or ammonium citrate or an organic solvent, such as ethanol or 2-ethyl-2, 4-pentanediol. However, such procedures do not provide a suitable means for producing crystalline human leukocyte interferons.
A versatile crystallizing agent is polyethylene glycol (PEG), which combines some of the characteristics of the salts and the organic solvents. See in this regard K. B. Ward et al., J. Mol. Biol. 98, 161(1975) and A. McPherson, Jr., J. Biol. Chem. 251, 6300 (1976). It has now been discovered that polyethylene glycol and particularly polyethylene glycol 4000 can be successfully used to crystallize the aforementioned human leuckocyte interferons, most particularly IFL-rA.
The human leukocyte interferons employed as starting materials in the instant crystallization process can be isolated by procedures providing the compounds in an essentially homogeneous state. Such procedures include high performance liquid chromatography (HPLC) such as described in the aforesaid U.S. Pat. No. 4,289,690, affinity chromatography utilizing a monoclonal antibody to human leukocyte interferon supported on a column support material as described, for example, by T. Staehelin et al., J. Biol. Chem. 256, 9750-9754 (1981) or any other procedure providing human interferon in sufficient purity (&gt;95%) and in sufficient concentration (.gtoreq.0.2 mg/ml interferon).
There are a number of important advantages attending to being able to obtain human leukocyte interferons in crystalline form. As indicated above one evident advantage is the additional purification available from crystallization step which could remove different impurities than the HPLC or conventional column chromatography can achieve. Moreover, crystalline human leukocyte can be stored and shipped at ambient temperatures free of the risk of protease contamination possible in solution storage. Other techniques for producing proteins such as lyophilization are known to cause some denaturation of interferon as evidenced by a loss in the value of the specific activity of samples before and after such procedures.
A suitable procedure for the crystallization of one of the human leukocyte interferons, IFL-rA, is set forth below in the Example. In analogous fashion the other human leukocyte interferons which exhibit a high degree of sequence homology can be crystallized.